Nanobody Research Articles

Lymphodepletion Enables Successful BCMA CAR-T Cell Engraftment and Tumour Control in the Syngeneic Vk*MYC Model of Aggressive Myeloma

DISCUSSION AND CONCLUSION Chimeric antigen receptor (CAR)-T cells directed against B-cell maturation antigen (BCMA), have yielded impressive results in clinical trials for multiply relapsed/refractory multiple myeloma (MM). However, progression-free survival is short, demonstrating a need for improvements. While numerous advances have been made to improve this treatment modality, these have not always translated to superior clinical outcomes. A major limitation is that novel therapeutics are often not tested in a relevant in vivo model. These novel strategies have been largely tested only in immunodeficient models of MM. To better recapitulate the host-tumour-effector cell interactions, particularly in the TME, we sought to develop a system to study CAR-T cell activity in the Vk*MYC model of MM. Here, we report on the first known successful model of CAR-T cell activity in an immunocompetent model of MM. We sought to identify a BCMA CAR construct that is capable of binding to both human and mouse BCMA. A novel nanobody recognizing BCMA was developed by immunizing llamas with human BCMA and the resulting single-domain antibodies (sdAbs) were subsequently isolated and cloned. These sdAbs were then screened for binding to mouse BCMA. A binder capable of recognizing both human and mouse BCMA was identified and cloned into our murine CAR construct, with CD28 and CD3z signaling domains, herein referred to as sdBCMA2.CD28.CD3z. Murine CAR-T cells were then generated by transducing activated healthy murine splenocytes with a retroviral vector carrying the sdBCMA2.CD28.CD3z construct. These CAR-T cells were then characterized in vitro using standard cytotoxicity, proliferation, and cytokine release assays. To assess activity in an immunocompetent model, female C57BL/6J mice were injected intravenously with Vk12598 Vk*MYC cells. CAR-T cells were later administered intravenously. Mice were bled weekly to quantify M-protein disease burden by serum protein electrophoresis and for phenotypic characterization of circulating cells. When co-cultured with BCMA+ human MM cell lines and with Vk*MYC cell lines, murine sdBCMA2.CD28.CD3z CAR-T cells effectively lysed target cells, proliferated, and released inflammatory cytokines. To assess the in vivo activity of these CAR-T cells, we injected intravenously female C57BL/6J mice with Vk12598 cells, and then administered sdBCMA2.CD28.CD3z CAR-T cells intravenously. While there was some evidence of survival improvement, the median overall survival (mOS) of 35 days compared to 28 days, for CAR-T vs. Mock T-cells, respectively, the difference was non-significant, and the survival benefit was not durable. To address this, we incorporated lymphodepletion by total body irradiation (TBI) followed by CAR-T cell administration the next day. By incorporating lymphodepletion prior to CAR-T cell administration we were able to significantly reduce M-protein surrogates of disease burden, as well as achieve durable remissions in the TBI + CAR-T arm with an mOS not yet reached after experimental day 91, compared to an mOS of 42 days in the TBI + Mock T-cell arm. The addition of TBI also significantly improved the engraftment of CAR-T cells, as assessed by flow cytometry. The TBI + CAR-T arm also had significantly increased IL-6, IL-1b, IL-2, and TNF-a in the serum, compared to the TBI + Mock arm, at 7 days post administration of CAR-T cells. The durable remission achieved using the combination of our novel sdBCMA2.CD28.CD3z CAR-T cells with lymphodepletion is a pivotal advancement in modelling MM CAR-T cell activity in this clinically relevant model system. Our approach represents the first time that CAR-T cells were successfully used to treat the Vk12598 model of MM that faithfully recapitulates relapsed MM. The incorporation of lymphodepletion significantly improves CAR-T cell engraftment, in line with clinical CAR-T cell therapy paradigms. The cytokine profile observed is consistent with the cytokine elevations typically observed after CAR-T cell administration in patients. As our construct is capable of binding to both human and mouse BCMA, we are therefore able to fully characterize our lead assets in a relevant model system. Our strategy will enable further research to dissect host-tumour-effector cell interactions, to better inform the design and characterization of novel CAR-T cell therapies in MM.

Enteric pharmacokinetics of monomeric and multimeric camelid nanobody single-domain antibodies.

Single-domain antibodies (sdAbs) derived from Camelidae heavy-chain-only antibodies (also called nanobodies or VHHs) have advantages over conventional antibodies in terms of their small size and stability to pH and temperature extremes, their ability to express well in microbial hosts, and to be functionally multimerized for enhanced properties. For these reasons, VHHs are showing promise as enteric disease therapeutics, yet little is known as to their pharmacokinetics (PK) within the digestive tract. To improve understanding of enteric VHH PK, we investigated the functional and structural stability of monomeric and multimeric camelid VHH-agents following in vitro incubation with intestinal extracts (chyme) from rabbits and pigs or fecal extracts from human sources, and in vivo in rabbits. The results showed that unstructured domains such as epitopic tags and flexible spacers composed of different amino acid sequences were rapidly degraded by enteric proteases while the functional core VHHs were much more stable to these treatments. Individual VHHs were widely variable in their functional stability to GI tract proteases. Some VHH-based agents which neutralize enteric Shiga toxin Stx2 displayed a functional stability to chyme incubations comparable to that of Stx2-neutralizing IgG and IgA mAbs, thus indicating that selected nanobodies can approach the functional stability of conventional immunoglobulins. Enteric PK data obtained from in vitro incubation studies were consistent with similar incubations performed in vivo in rabbit surgical gut loops. These findings have broad implications for enteric use of VHH-based agents, particularly VHH fusion proteins.

A novel single domain bispecific antibody targeting VEGF and TNF-α ameliorates rheumatoid arthritis

Anti-TNF-α therapy fails in 30% of patients, where TNF-α may not be the key causative factor in these patients. We developed a bispecific single-domain antibody block TNF-α and VEGF (V5-3).The experiments showed that V5-3 effectively activated proliferation and migration of RA-FLS and HUVEC, tube-forming role of HUVEC, and expression of inflammatory factors in vitro. Besides, the experiments indicated that the anti-RA activity of V5-3 was superior to Anbainuo in vivo. Application of V5-3 reduced the expression of inflammatory factors, extent of synovial inflammation and angiogenesis and attenuated the severity of autoimmune arthritis in collagen-induced arthritis (CIA) mice. Mechanistically, V5-3 suppressed p65, AKT and VEGFR2 phosphorylation, as well as production of TNF-α and VEGF in joint tissues. These results demonstrated that V5-3 displayed a superior effect of anti-RA, may be a new therapy to overcome the limitations of anti-TNF-α monoclonal antibody.

Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus.

Arming oncolytic viruses with transgenes encoding immunomodulators improves their therapeutic efficacy by enhancing and/or sustaining the innate and adaptive anti-tumoral immune responses. We report here the isolation, selection, and vectorization of a blocking anti-human PDL1 single-domain antibody (sdAb) isolated from PDL1-immunized alpacas. Several formats of this sdAb were vectorized into the vaccinia virus (VV) and evaluated for their programmed cell death protein 1 (PD1)/PD1 ligand (PDL1) blocking activity in the culture medium of tumor cells infected in vitro. In those conditions, VV-encoded homodimeric sdAb generated superior PDL1 blocking activity compared to a benchmark virus encoding full-length avelumab. The sdAb was further used to design simple, secreted, and small tumor necrosis factor superfamily (TNFSF) fusions with the ability to engage their cognate receptors (TNFRSF) only in the presence of PDL1-positive cells. Finally, PDL1-independent alternatives of TNFRSF agonists were also constructed by fusing different variants of surfactant protein-D (SP-D) oligomerization domains with TNFSF ectodomains. An optimal SP-D-CD40L fusion with an SP-D collagen domain reduced by 80% was identified by screening with a transfection/infection method where poxvirus transfer plasmids and vaccinia virus were successively introduced into the same cell. However, once vectorized in VV, this construct had a much lower CD40 agonist activity compared to the SP-D-CD40L construct, which is completely devoid of the collagen domain that was finally selected. This latest result highlights the importance of working with recombinant viruses early in the payload selection process. Altogether, these results bring several complementary solutions to arm oncolytic vectors with powerful immunomodulators to improve their immune-based anti-tumoral activity.

The GEM-handle as convenient labeling strategy for bimodal single-domain antibody-based tracers carrying 99mTc and a near-infrared fluorescent dye for intra-operative decision-making.

Intra-operative fluorescence imaging has demonstrated its ability to improve tumor lesion identification. However, the limited tissue penetration of the fluorescent signals hinders the detection of deep-lying or occult lesions. Integrating fluorescence imaging with SPECT and/or intra-operative gamma-probing synergistically combines the deep tissue penetration of gamma rays for tumor localization with the precision of fluorescence imaging for precise tumor resection. In this study, we detail the use of a genetically encoded multifunctional handle, henceforth referred to as a GEM-handle, for the development of fluorescent/radioactive bimodal single-domain antibody (sdAb)-based tracers. A sdAb that targets the urokinase plasminogen activator receptor (uPAR) was engineered to carry a GEM-handle containing a carboxy-terminal hexahistidine-tag and cysteine-tag. A two-step labeling strategy was optimized and applied to site-specifically label IRDye800CW and 99mTc to the sdAb. Bimodal labeling of the sdAbs proved straightforward and successful. 99mTc activity was however restricted to 18.5 MBq per nmol fluorescently-labeled sdAb to prevent radiobleaching of IRDye800CW without impeding SPECT/CT imaging. Subsequently, the in vivo biodistribution and tumor-targeting capacity of the bimodal tracer were evaluated in uPAR-positive tumor-bearing mice using SPECT/CT and fluorescence imaging. The bimodal sdAb showed expected renal background signals due to tracer clearance, along with slightly elevated non-specific liver signals. Four hours post-injection, both SPECT/CT and fluorescent images achieved satisfactory tumor uptake and contrast, with significantly higher values observed for the anti-uPAR bimodal sdAb compared to a control non-targeting sdAb. In conclusion, the GEM-handle is a convenient method for designing and producing bimodal sdAb-based tracers with adequate in vivo characteristics.

A specific nanobody-based affinity chromatography resin as a platform for small ubiquitin-related modifier fusion protein purification

As an excellent fusion tag for expressing heterologous proteins, yeast SUMO (small ubiquitin-related modifier) has unique advantages such as improving solubility, promoting stability, and reducing degradation, but it lacks a simple and rapid purification method. Camelid single-domain antibodies (VHHs or nanobodies) show great promise as an efficient tool in analytical application. In this study, VHHs against SUMO protein were isolated for the first time using biopanning of an immune camelid nanobody library. Among these nanobodies, VS2 demonstrated a high expression level (1.12 g L − 1), and a high affinity for SUMO (2.26 nM). Meanwhile, VHHs were coupled to agarose resins by cysteine at the C-terminal to form affinity chromatography resins. The VS2 resin showed excellent specificity and a dynamic binding capacity for SUMO, SUMO-DsbA (disulfide oxidoreductase) and SUMO-SAM (S-adenosylmethionine synthetase) were 2.41 mg/mL resin, 7.57 mg/mL resin and 16.23 mg/mL resin, respectively. Furthermore, the VS2 resin enabled one-step purification of SUMO-fusions [SUMO-Fc (human IgG1-Fc fragment), SUMO-IGF1 (human insulin-like growth factor 1), SUMO-FGF21 (human fibroblast growth factor 21), SUMO-G-CSF (human Granulocyte colony-stimulating factor), SUMO-PDGF (human platelet-derived growth factor) and SUMO-PAS200 (conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala-and Ser)], and maintained binding capacity and selectivity over 25 purification cycles, each including 15 min of cleaning-in-place with 0.1 M NaOH. This study demonstrated that the VS2 resin was a useful tool at the laboratory scale for one-step purification of various SUMO fusions from complex mixtures.

Humanized single-domain antibody targeting HER2 enhances function of chimeric antigen receptor T cells.

Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors, and each component plays an important role in the function and anti-tumor efficacy. It has been reported that using human sequences or a low affinity of CAR single-chain variable fragments (scFvs) in the CAR binding domains is a potential way to enhance the function of CAR-T cells. However, it remains largely unknown how a lower affinity of CARs using humanized scFvs affects the function of CAR-T cells until recently. We used different humanized anti-HER2 antibodies as the extracellular domain of CARs and further constructed a series of the CAR-T cells with different affinity. We have observed that moderately reducing the affinity of CARs (light chain variable domain (VL)-based CAR-T) could maintain the anti-tumor efficacy, and improved the safety of CAR therapy both in vitro and in vivo compared with high-affinity CAR-T cells. Moreover, T cells expressing the VL domain only antibody exhibited long-lasting tumor elimination capability after multiple challenges in vitro, longer persistence and lower cytokine levels in vivo. Our findings provide an alternative option for CAR-T optimization with the potential to widen the use of CAR T cells.

A TRIM21-based bioPROTAC highlights the therapeutic benefit of HuR degradation

Human antigen R (HuR) is a ubiquitously expressed RNA-binding protein, which functions as an RNA regulator. Overexpression of HuR correlates with high grade tumours and poor patient prognosis, implicating it as an attractive therapeutic target. However, an effective small molecule antagonist to HuR for clinical use remains elusive. Here, a single domain antibody (VHH) that binds HuR with low nanomolar affinity was identified and shown to inhibit HuR binding to RNA. This VHH was used to engineer a TRIM21-based biological PROTAC (bioPROTAC) that could degrade endogenous HuR. Significantly, HuR degradation reverses the tumour-promoting properties of cancer cells in vivo by altering the HuR-regulated proteome, highlighting the benefit of HuR degradation and paving the way for the development of HuR-degrading therapeutics. These observations have broader implications for degrading intractable therapeutic targets, with bioPROTACs presenting a unique opportunity to explore targeted-protein degradation through a modular approach.

CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models

BackgroundRhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, whose prognosis is still poor especially for metastatic, high-grade, and relapsed RMS. New treatments are urgently needed, especially systemic therapies. Chimeric Antigen Receptor T cells (CAR Ts) are very effective against hematological malignancies, but their efficacy against solid tumors needs to be improved. CD276 (B7-H3) is a target upregulated in RMS and detected at low levels in normal tissues. FGFR4 is a very specific target for RMS. Here, we optimized CAR Ts for these two targets, alone or in combination, and tested their anti-tumor activity in vitro and in vivo.MethodsFour different single-domain antibodies were used to select the most specific FGFR4-CAR construct. RMS cell killing and cytokine production by CD276- and FGFR4-CAR Ts expressing CD8α or CD28 HD/TM domains in combination with 4-1BB and/or CD28 co-stimulatory domains were tested in vitro. The most effective CD276- and FGFR4-CAR Ts were used to generate Dual-CAR Ts. Tumor killing was evaluated in vivo in three orthotopic RMS mouse models.ResultsCD276.V-CAR Ts (276.MG.CD28HD/TM.CD28CSD.3ζ) showed the strongest killing of RMS cells, and the highest release of IFN-γ and Granzyme B in vitro. FGFR4.V-CAR Ts (F8-FR4.CD28HD/TM.CD28CSD.3ζ) showed the most specific killing. CD276-CAR Ts successfully eradicated RD- and Rh4-derived RMS tumors in vivo, achieving complete remission in 3/5 and 5/5 mice, respectively. In CD276low JR-tumors, however, they achieved complete remission in only 1/5 mice. FGFR4 CAR Ts instead delayed Rh4 tumor growth. Dual-CAR Ts promoted Rh4-tumors clearance in 5/5 mice.ConclusionsCD276- and CD276/FGFR4-directed CAR Ts showed effective RMS cell killing in vitro and eradication of CD276high RMS tumors in vivo. CD276low tumors escaped the therapy highlighting a correlation between antigen density and effectiveness. FGFR4-CAR Ts showed specific killing in vitro but could only delay RMS growth in vivo. Our results demonstrate that combined expression of CD276-CAR with other CAR does not reduce its benefit. Introducing immunotherapy with CD276-CAR Ts in RMS seems to be feasible and promising, although CAR constructs design and target combinations have to be further improved to eradicate tumors with low target expression.

In vitro modelling of local gene therapy with IL-15/IL-15Rα and a PD-L1 antagonist in melanoma reveals an interplay between NK cells and CD4+ T cells

Blockade of the immune checkpoint axis consisting of programmed death-1 (PD-1) and its ligand PD-L1 alleviates the functional inhibition of tumor-infiltrating lymphoid cells yet weakly induces their expansion. Exogenous cytokines could further expand lymphoid cells and thus synergize with αPD-L1 therapy. However, systemic delivery of most cytokines causes severe toxicity due to unspecific expansion of immune cells in the periphery. Here, we modelled local delivery of cytokines and αPD-L1 therapeutics to immune cell-containing in vitro melanoma tumors. Three-dimensional tumor models consisting of 624-MEL cells were co-cultured with human peripheral blood lymphoid cells (PBLs) in presence of the cytokines IL-2, IL-7, IL-15, IL-21 and IFN-γ. To model local gene therapy, melanoma tumors were modified with lentiviral vectors encoding IL-15 fused to IL-15Rα (IL-15/IL-15Rα) and K2-Fc, a fusion of a human PD-L1 specific single domain antibody to immunoglobulin (Ig)G1 Fc. To evaluate the interplay between PBL fractions, NK cells, CD4+ T cells or CD8+ T cells were depleted. Tumor cell killing was followed up using real time imaging and immune cell expansion and activation was evaluated with flow cytometry. Among the tested cytokines, IL-15 was the most potent cytokine in stimulating tumor cell killing and expanding both natural killer (NK) cells and CD8+ T cells. Gene-based delivery of IL-15/IL-15Rα to tumor cells, shows expansion of NK cells, activation of NK cells, CD4+ and CD8+ T cells, and killing of tumor spheroids. Both NK cells and CD8+ T cells are necessary for tumor cell killing and CD4+ T-cell activation was reduced without NK cells. Co-delivery of K2-Fc improved tumor cell killing coinciding with increased activation of NK cells, which was independent of bystander T cells. CD4+ or CD8+ T cells were not affected by the co-delivery of K2-Fc even though NK-cell activation impacted CD4+ T-cell activation. This study demonstrates that gene-based delivery of IL-15/IL-15Rα to tumor cells effectively mediates anti-tumor activity and sensitizes the tumor microenvironment for therapy with αPD-L1 therapeutics mainly by impacting NK cells. These findings warrant further investigation of gene-based IL-15 and K2-Fc delivery in vivo.

Human Single Domain Antibody-Based CAR-T Cells Targeting BAFF-R Demonstrate Promising Preclinical Activity in B Cell Malignancies

Background: B-cell activating factor-receptor (BAFF-R) is a potential B-cell specific target, highly expressed in B cell malignancies and regulates B cell proliferation and survival. Single-chain antibody-based CAR-T cells targeting BAFF-R (PMB101) has demonstrated antitumor effects against human B cell malignancies and can overcome CD19 antigen loss (Qin et al. Sci Transl Med. 2019;11(511)). However, CARs built on antigen-specific single chain antibody variable fragment (scFv) may have some immunological risks. Recently, single variable domain of heavy chain antibodies are becoming an alternative to scFv to construct CARs. To improve the function of BAFF-R CAR-T cells, we designed CARs with only a fully human heavy-chain variable domain. Methods: A fully human antibody phage display library (IMARS, IASO Biotherapeutics) was used to select anti-BAFF-R clones using optimal protein/cell alternate panning. The enzyme-linked immunosorbent assay (ELISA) and flow cytometry were applied to evaluate the specificity of clones. The screened clones were grafted into a second-generation CAR with CD8α hinge, 4-1BB costimulatory and CD3ζ intracellular signaling domains to generate CAR-T cells. PMB101 and CAR-T cells constructed with VAY736 were used as positive controls. Non-transduced T cells from the same donor were used as a negative control. Results: Four candidate clones including clone 1, 5, 77 and 80 were finally screened by in vitro functional evaluation, among which clone 5, 77 and 80 were variable domain heavy chain antibodies. The expression levels of the degranulation marker CD107a on CAR-T cells constructed with the four candidate clones were significantly upregulated after coculture with BAFF-R positive tumor cells, while BAFF-R negative tumor cells could not effectively activate CAR-T cells (data not shown). The four clones exhibited similar cytotoxicity in CAR-T cells compared with the PMB101 and VAY736 CAR-T cells using the luciferase-based cytotoxicity assay (data not shown). Xenografts were established in NPG mice following intravenous injection of 1 x 10 6 jeko-1 cells expressing the firefly luciferase gene on day - 5, and a single dose of 4 x 10 6 CAR T cells were infused IV on day 0. Compared to the other groups, no progressive tumor growth was discovered in mice treated with clone 5 CAR-T cells until day 49. Clone 5 CAR-T cells showed excellent in vivo antitumor activity to PMB101 and conferred long-term survival (data not shown). Conclusion: We developed a novel BAFF-R CAR-T cell product structured on single-domain antibody. This product demonstrated promising preclinical activity, and it may provide an alternative choice for patients with B cell malignancies.

O-72 - Site-specifically THP-functionalized hPD-L1 single-domain antibody: preliminary data toward kit development

O-72 - Site-specifically THP-functionalized hPD-L1 single-domain antibody: preliminary data toward kit development

Preparation and Crystallographic Analysis of a Complex of SARS-CoV-2 S-Protein Receptor-Binding Domain with a Virus-Neutralizing Nanoantibody

Coronavirus SARS-CoV-2 was the cause of global pandemic COVID-19 that has claimed millions of lives around the world. One of the approaches to the therapy of severe forms of COVID-19 is based on the use of virus-neutralizing antibodies against the receptor-binding domain (RBD) of the virus spike glycoprotein (S). Detailed structural data on particular epitopes recognized by these virus-neutralizing antibodies is an important aspect of the improvement of available antibodies in order to extend the range of recognized virus strains. A stoichiometric complex of the virus-neutralizing single-domain antibody P2C5 and the glycosylated form of SARS-CoV-2 S protein RBD was obtained. Despite numerous conditions in which the crystal growth of the complex was observed, these crystals showed poor diffraction, apparently because of heterogeneity associated with the glycosylation of RBD. With the aim of improving the crystal quality, deglycosylase PNGase F was produced in a bacterial expression system and deglycosylation of RBD was performed. Despite low solubility of the deglycosylated form of RBD, it retains the ability to bind to the nanoantibody P2C5, resulting in the formation of a stable heterodimeric complex during gel filtration. The crystals of this complex were obtained. They showed diffraction to a similar resolution of ~4 Å on a laboratory X-ray diffractometer and at a synchrotron radiation source. The symmetry space group, the unit cell parameters of the crystal, and its protein composition were determined.

O-58 - Development of [68Ga]Ga-DOTA-4AH29, a radiolabeled single-domain antibody for PET imaging of FAP-α expression in cancer

O-58 - Development of [68Ga]Ga-DOTA-4AH29, a radiolabeled single-domain antibody for PET imaging of FAP-α expression in cancer

P-233 - Preclinical characterization of [18F]FB-labeled single-domain antibodies for PET imaging of FAP-α or Folate-α overexpression in cancer

P-233 - Preclinical characterization of [18F]FB-labeled single-domain antibodies for PET imaging of FAP-α or Folate-α overexpression in cancer

Mirror-Image Single-Domain Antibody for a Novel Nonimmunogenic Drug Scaffold.

Immunogenic responses by protein therapeutics often lead to reduced therapeutic effects and/or adverse effects via the generation of neutralizing antibodies and/or antidrug antibodies (ADA). Mirror-image proteins of the variable domain of the heavy chain of the heavy chain antibody (VHH) are potential novel protein therapeutics with high-affinity binding to target proteins and reduced immunogenicity because these mirror-image VHHs (d-VHHs) are less susceptible to proteolytic degradation in antigen-presenting cells (APCs). In this study, we investigated the preparation protocols of d-VHHs and their biological properties, including stereoselective target binding and immunogenicity. Initially, we established a facile synthetic process of two model VHHs [anti-GFP VHH and PMP12A2h1 (monomeric VHH of caplacizumab)] and their mirror-image proteins by three-step native chemical ligations (NCLs) from four peptide segments. The folded synthetic VHHs (l-anti-GFP VHH and l-PMP12A2h1) bound to the target proteins (EGFP and vWF-A1 domain, respectively), while their mirror-image proteins (d-anti-GFP VHH and d-PMP12A2h1) showed no binding to the native proteins. For biodistribution studies, l-VHH and d-VHH with single radioactive indium diethylenetriamine-pentaacid (111In-DTPA) labeling at the C-terminus were designed and synthesized by the established protocol. The distribution profiles were essentially similar between l-VHH and d-VHH, in which the probes accumulated in the kidney within 15 min after intravenous administration in mice, because of the small molecular size of VHHs. Comparative assessment of the immunogenicity responses revealed that d-VHH-induced levels of ADA generation were significantly lower than those of native VHH, regardless of the peptide sequences and administration routes. The resulting scaffold investigated should be applicable in the design of d-VHHs with various C-terminal CDR3 sequences, which can be identified by screening using display technologies.

Novel Approach for Obtaining Variable Domain of New Antigen Receptor with Different Physicochemical Properties from Japanese Topeshark (Hemitriakis japanica).

Diverse candidate antibodies are needed to successfully identify therapeutic and diagnostic applications. The variable domain of IgNAR (VNAR), a shark single-domain antibody, has attracted attention owing to its favorable physicochemical properties. The phage display method used to screen for optimal VNARs loses sequence diversity because of the bias caused by the differential ease of protein expression in Escherichia coli. Here, we investigated a VNAR selection method that combined panning with various selection pressures and next-generation sequencing (NGS) analyses to obtain additional candidates. Drawing inspiration from the physiological conditions of sharks and the physicochemical properties of VNARs, we examined the effects of NaCl and urea concentrations, low temperature, and preheating at the binding step of panning. VNAR phage libraries generated from Japanese topeshark (Hemitriakis japanica) were enriched under these conditions. We then performed NGS analysis and attempted to select clones that were specifically enriched under each panning condition. The identified VNARs exhibited higher reactivity than those obtained by panning without selection pressure. Additionally, they possess physicochemical properties that reflect their respective selection pressures. These results can greatly enhance our understanding of VNAR properties and offer guidance for the screening of high-quality VNAR clones that are present at low frequencies.

Yeast Surface Dual Display Platform for Rapid Discovery of Shark VNAR from a Semi-Synthetic Library Followed by Next-Generation Sequencing

The shark-derived single-domain antibody VNAR (variable domain of new antigen receptor) has many advantageous features that make the VNAR suitable for improving current monoclonal antibody therapy deficiencies or disease diagnosis methods. In order to discover more VNARs, it is necessary to improve the efficiency of the isolation process. This research aims to enhance the VNAR discovery platform by dual displaying the semi-synthetic VNAR library and green fluorescent protein tag on the yeast surface. The GFP tag can be used to determine the degree of VNAR expression. The diversity of the semi-synthetic VNAR library constructed in this study is verified to be 1.97 × 109 by next-generation sequencing (NGS). We conveniently screened VNARs against the feline neonatal Fc receptor or feline infectious peritonitis virus nucleocapsid protein by sequential MACS and FACS. To find more diverse VNARs, we analyzed the NGS data of VNAR CDR3 genes before and after biopanning. By comparing the frequency change of each sequence, we found that the amplification factor of sequences was increased by biopanning. Four VNAR candidates selected by the high-frequency and high-amplification factor criteria showed an antigen-binding ability. The results demonstrate that biopanning from a yeast surface displaying a semi-synthetic VNAR library followed by the NGS assay can generate antigen binders rapidly without the need for shark rearing and long-term immunization.

Discovery of Nanosota-2, -3, and -4 as super potent and broad-spectrum therapeutic nanobody candidates against COVID-19.

The COVID-19 pandemic exposed limitations of conventional antibodies as therapeutics, including high cost, limited potency, ineffectiveness against new viral variants, and primary reliance on injection-only delivery. Nanobodies are single-domain antibodies with therapeutic potentials. We discovered three anti-SARS-CoV-2 nanobodies, named Nanosota-2, -3, and -4, from an immunized alpaca. Nanosota-2 is super potent against prototypic SARS-CoV-2, Nanosota-3 is highly potent against the omicron variant, and Nanosota-4 is effective against both SARS-CoV-1 and SARS-CoV-2. In addition to their super potency and combined broad antiviral spectrum, these nanobodies are cost-effective, can be easily adapted to new viral variants through phage display, and can potentially be administered as inhalers. The Nanosota series are powerful therapeutic candidates to combat circulating SARS-CoV-2 and prepare for possible future coronavirus pandemics.

Site-Specific Modification of Single Domain Antibodies by Enzyme-Immobilized Magnetic Beads.

Nanobodies as imaging agents and drug conjugates have shown great potential for cancer diagnostics and therapeutics. However, site-specific modification of a nanobody with microbial transglutaminase (mTGase) encounters problems in protein separation and purification. Here, we describe a facile yet reliable strategy of immobilizing mTGase onto magnetic beads for site-specific nanobody modification. The mTGase immobilized on magnetic beads (MB-mTGase) exhibits catalytic activity nearly equivalent to that of the free mTGase, with good reusability and universality. Magnetic separation simplifies the protein purification step and reduces the loss of nanobody bioconjugates more effectively than size exclusion chromatography. Using MB-mTGase, we demonstrate site-specific conjugation of nanobodies with fluorescent dyes and polyethylene glycol molecules, enabling targeted immunofluorescence imaging and improved circulation dynamics and tumor accumulation in vivo. The combined advantages of MB-mTGase method, including high conjugation efficiency, quick purification, less protein loss, and recycling use, are promising for site-specific nanobody functionalization and biomedical applications.